PubMed Health⌕ Search

Biomedical subjects

N S Chang

Publications and source records attributed to N S Chang.

14 recordsLinked to original sources
PubMed1995

Transforming growth factor-beta 1 induction of novel extracellular matrix proteins that trigger resistance to tumor necrosis factor cytotoxicity in murine L929 fibroblasts.

The molecular basis by which transforming growth factor (TGF)-beta 1 protects certain tumor cells from tumor necrosis factor (TNF) cytotoxicity was investigated. When pretreated, with TGF-beta 1, -beta 2, and -beta 3, murine L929S fibroblasts developed resistance to TNF cytotoxicity. Time course experiments revealed that TGF-beta 1 initially induced both cellular protein-tyrosine phosphorylation and simultaneous secretion of a novel extracellular matrix TNF-resistance triggering (TRT) protein(s), which closely preceded the acquisition of TNF-resistance. TGF-beta 2 and -beta 3 also increased tyrosine phosphorylation. However, both molecules failed to stimulate TRT secretion. The increased levels of phosphorylation, particularly to 9 specific protein tyrosine kinase inhibitor-sensitive cellular proteins, appeared to alter the TNF killing pathway. TGF-beta 1-induced TRT secretion required participation of unknown serum factors. TRT adhered strongly to polystyrene plates and resisted treatment with heat (60 degrees C, 30 min), collagenase, alpha 2-macroglobulin, heparin, antibodies against TGF-beta s, and limited trypsin digestion. Notably, TRT promoted TNF-resistance via activation of tyrosine and serine/threonine kinase functions in L929S. Thus, the molecular pathway involves TGF-beta 1-mediated initiation of a rapid tyrosine phosphorylation of cellular protein substrates (which alters TNF cytotoxic pathway), and a simultaneous secretion of TRT, which in turn signals the cells to maintain the levels of phosphorylation, thereby sustaining the TNF-resistance.

Alkaloids↗
PubMed1995

Role of peptide hydrophilicity on determination of microsequencing efficiency.

The successful sequencing of short peptides on hydrophobic polyvinylidene difluoride membrane (PVDF-P) has been problematic. In this study the sequencing efficiency of various short synthetic peptides on charged-modified PVDF (PVDF-N) and chemically treated glass-fiber membranes or discs has been examined. These modified membranes provided better repetitive yields or sequencing efficiency than the unmodified PVDF-P. In contrast, there were no significant differences among the resulting initial yields for all the tested membranes, indicating that the modified membranes did not interfere with the coupling/cleaving reactions. Methanol at 1% increased the solubility of phenylisothiocyanate (PITC) in heptane for gas-phase delivery during the coupling reaction, whereas this addition of methanol failed to increase the coupling efficiency. Reduction of chemical background noise by replacing triethylamine (TEA) with diisopropylethylamine (DIPEA) also failed to increase the coupling efficiency. Polybrene strengthened the peptide binding to both PVDF-P and PVDF-N, but increased the amount of carry-over of PTH-amino acid from the current cycle to the next. Nonetheless, hydrophilic peptides had higher sequencing recoveries and repetitive yields than hydrophobic peptides when sequenced on all the tested membranes. This relationship was further verified by testing a synthetic peptide with decreasing hydrophilicity by sequential deletions of 2 amino acid residues from its N-terminus. A decreasing sequencing efficiency was observed, which correlated with the reduced hydrophilicity and peptide length. Similar results were obtained when testing peptide fragments with decreasing hydrophilicity by deletions of amino acids from the C-terminus.(ABSTRACT TRUNCATED AT 250 WORDS)

Amines↗
PubMed1994

Synthetic polysulfated hyaluronic acid is a potent inhibitor for tumor necrosis factor production.

Based on the premise that naturally occurring glycosaminoglycans could serve as building blocks for synthesizing nontoxic drugs for suppression of tumor necrosis factor (TNF) production by inflammatory cells, we have chemically modified hyaluronic acid (HA) and tested its effects in blocking TNF-alpha and TNF-beta production in vitro. HA was chosen mainly for its structural simplicity, nonimmunogenicity, and readiness for chemical modifications. When HA was chemically polysulfated to a sulfate/hexosamine molar ratio of 3.9, the sulfated HAs was shown to be a potent inhibitor of TNF-alpha production in lipopolysaccharide (LPS)- or interferon-gamma-activated THP-1 cells. For example, a concentration of HAs as low as 10 ng/ml reduced TNF-alpha production in LPS-activated THP-1 cells more than 50%, whereas achieving a similar extent of reduction required 50 micrograms/ml native HA. By decreasing the extent of polysulfation, the inhibitory effect of HAs on TNF-alpha production was diminished. Other chemical modifications, including deacetylation, thiolation, or reduction of the carboxylic groups, could not increase the efficacy of HA in suppression of TNF-alpha production. Naturally polysulfated glycosaminoglycans, such as chondroitin sulfates, keratan sulfate, heparan sulfate, and heparin, failed to inhibit TNF-alpha production. HAs also restricted TNF-beta (lymphotoxin) secretion in an Epstein-Barr virus-transformed B cell line, Roha-9, which constitutively produces TNF-beta. HAs had no inhibitory effect on the proliferation of THP-1 or Roha-9 cells, which would account for the reduced TNF-alpha or TNF-beta production. Furthermore, time-course metabolic labeling studies revealed that HAs could not restrict overall protein synthesis and secretion in THP-1 cells. However, HAs increased complement C1q secretion in THP-1 in a dose-dependent manner, but it had no effect on biosynthesis of complement C1 inhibitor, factor D, and Fc gamma receptor type II (Fc gamma RII). These results indicate that HA, selectively restricts the production of TNF-alpha, TNF-beta, and probably several other protein species.

Cell Division↗
PubMed1994

Identification of a 46-kD latex protein allergen in health care workers.

Latex allergy is an occupational hazard for health care workers. Extractable latex proteins are known to be allergenic, but most latex allergens have not been specifically identified. The purpose of this study was to characterize the IgE response of latex-allergic patients to latex proteins and to identify common protein allergens. Serum was obtained from 40 individuals who were skin test-positive to latex; 85% were health care workers. Western blots for IgE reactivity were performed using both ammoniated (AL) and non-ammoniated (NAL) latex proteins and IgE-reactive NAL proteins were analysed by microsequence analysis. The patients were grouped according to common patterns of reactivity. Pattern 1, the most common pattern of reactivity (9/40 patients) recognized two protein bands in both NAL and AL at 46 and 110 kD. A second, heterogeneous pattern of reactivity (pattern 2) recognized a diffuse pattern of polypeptides in the AL preparation. The n-terminal amino acid sequences for allergens at 14, 18, 29, 46 and 110 kD were determined. Sequence analysis identified the 14-kD and 18-kD allergens as the hevein proprotein. The 46-kD and 110-kD had identical sequences which were unique from known latex proteins. We conclude that multiple latex proteins are allergens with hevein preprotein and a previously unidentified 46/110-kD protein being commonly recognized in health care workers.

Adolescent↗
PubMed1994

Role of N-terminal domain of histidine-rich glycoprotein in modulation of macrophage Fc gamma receptor-mediated phagocytosis.

A brief exposure of murine peritoneal inflammatory macrophages to plasma histidine-rich glycoprotein (HRG; 77,000-81,000 MW) for 1-2 hr increased Fc gamma receptor (Fc gamma R) expression and phagocytic function in these cells. However, a continual culture of the cells without the presence of HRG for the next 18-48 hr resulted in down-regulation of Fc gamma R expression and phagocytic function. Similarly, HRG decreased Fc gamma RII expression in less differentiated human THP-1 monocytic cells during treatment for 18 hr, as determined by cellular ELISA and metabolic labelling. The molecular mechanism by which HRG regulates Fc gamma R expression is unknown. However, at a relatively high concentration (> 1 microgram/ml), HRG altered the cellular metabolism by increasing cellular protein synthesis but reducing protein secretion. These observations suggest a likely mechanism for the HRG-mediated reduction of Fc gamma R expression. A degraded HRG (40,000 MW) which possessed an identical N-terminal sequence as that of the native HRG was capable of decreasing macrophage Fc gamma R expression and phagocytosis. The results indicate that the functional domain of HRG responsible for binding to macrophages is localized to the N-terminal half.

Animals↗
PubMed1992

Regulation of complement functional efficiency by histidine-rich glycoprotein.

The modulation of complement functional efficiency by serum histidine-rich glycoprotein (HRG) was investigated. Addition of exogenous HRG to prewarmed diluted serum, followed immediately by sensitized sheep erythrocytes (EA), resulted in enhanced hemolysis. However, when HRG was incubated with diluted serum for 10 minutes at 37 degrees C, inhibition of hemolysis occurred. The biphasic modulation of complement function was also obtained with the complement alternative pathway when HRG was added to diluted serum for hemolysis of rabbit erythrocytes. Partial reduction of complement functional activity was shown when serum was absorbed by an HRG-Sepharose 6MB column. Western blot analysis showed that complement C8, C9, factor D, and S-protein in diluted serum were bound by nylon membrane-immobilized HRG. However, by immunoprecipitation of relatively undiluted serum with anti-HRG IgG beads, HRG was found to coprecipitate with S-protein and plasminogen, which suggested that HRG may complex with these proteins in serum. In functional tests, HRG inhibited C8 hemolytic activity, probably by preventing C8 binding to EAC1-7 cells. HRG also enhanced polymerization of purified C9 as well as the generation of a 45-Kd C9 fragment. Such an effect was even more pronounced in the presence of divalent cations with the reaction mixtures of C9 and HRG. Partial dimerization of C9 was shown when exogenous HRG was added to normal serum. In contrast, polymerization of serum C9 was inhibited by exogenous HRG during poly I:C activation of serum or incubation under low ionic strength conditions. HRG was further shown to inhibit factor D-mediated cleavage of factor B when bound by cobra venom factor. The molecular basis by which HRG regulates serum complement function is not clear. Hypothetically, the tandem repetitions of a consensus histidine-rich penta-peptide sequence in HRG may provide a highly charged area that interacts with complement components.

Animals↗
PubMed1992

Regulation of macrophage Fc receptor expression and phagocytosis by histidine-rich glycoprotein.

Regulation of macrophage Fc receptor (Fc gamma R)-mediated phagocytic function by histidine-rich glycoprotein (HRG) was investigated. Pretreatment of oil-elicited inflammatory mouse peritoneal macrophages with HRG for 1-3 hr increased their Fc gamma R-mediated binding and phagocytosis of IgG-opsonized sheep erythrocyte conjugates (EA). A significant reduction of Fc gamma R-dependent EA binding and phagocytosis occurred after pretreatment of macrophages with HRG for more than 8 hr. These results indicate that HRG is capable of modulating Fc gamma R expression in a biphasic fashion, which directly affects the overall efficiency of phagocytosis. HRG differentially regulated the functions of Fc gamma R subclasses. For example, HRG reduced the efficiency of Fc gamma RII (Fc gamma 2b/gamma 1R)-dependent phagocytosis of erythrocytes conjugated with monoclonal IgG2b or IgG1 by macrophages pretreated with HRG for 24 hr. However, when similar studies were performed using erythrocytes coated with monoclonal IgG2a, HRG was less effective in inhibiting Fc gamma RI (Fc gamma 2aR)-dependent phagocytosis. As an HRG-binding glycosaminoglycan, heparin failed to block the regulatory function of HRG on macrophages. Similarly, interferon-gamma (IFN-gamma) was not capable of blocking the functional activity of HRG. These studies suggest that HRG regulates macrophage function via a novel pathway different from that of heparin or IFN-gamma.

Animals↗
PubMed1991

Characterization of C1 inhibitor binding to neutrophils.

In a previous study we have isolated neutrophil membrane proteins that non-covalently bind to native C1-INH (105,000 MW) and a non-functional, degraded C1-INH (88,000 MW; C1-INH-88). To further characterize the binding nature, we have designed a novel kinetic C1 titration assay which enables not only a quantification of the removal of fluid-phase C1-INH by neutrophils, but also a concomitant measure of residual C1-INH function. Native C1-INH, when adsorbed to EDTA-pretreated neutrophils, lost its function in the inhibition of fluid-phase C1. The non-functional C1-INH-88, which is probably devoid of a reactive centre, was found to block the binding of native C1-INH to neutrophils. Pretreatment of neutrophils with serine esterase inhibitors did not abrogate binding capacity of the cells for C1-INH, whereas the binding affinity for C1-INH was lost when the cells were pretreated with trypsin. An array of human peripheral blood leucocytes and several lymphoid cell lines has surface binding sites for C1-INH, but not on human erythrocytes and U937 cells. Binding was further confirmed using (i) C1-INH-microsphere beads to neutrophils, in which the binding was blocked when pretreating neutrophils with excess C1-INH or with trypsin, and (ii) radiolabelled C1-INH to neutrophils, which was competitively blocked by unlabelled non-functional C1-INH-88. Desialylation of C1-INH significantly reduced its binding affinity for neutrophils, indicating that the membrane receptor sites on neutrophils could be specific for the binding of sialic acid residues on C1-INH. Overall, our studies indicate that neutrophils or other leucocytes possess specific surface binding sites for the sialic acid-containing portion of C1-INH.

Binding, Competitive↗
PubMed1986

Turbidimetric microassay for macrophage-mediated antibody-dependent cellular cytotoxicity.

An improved microassay for quantitation of murine macrophage-mediated antibody-dependent cellular cytotoxicity (ADCC) has been developed. The method is based on the turbidimetric measurement of sheep erythrocyte or nucleated (L1210) target cell suspensions at 630 nm with an automatic microtiter plate densitometer. The novel method was applied to demonstrate dose-related increases in murine macrophage mediated ADCC with varying antibody concentration, effector:target ratio, and incubation time. Advantages of the turbidimetric method were shown over the 51Cr-labeled target cell method by direct comparisons in that the new method was 2-4 times more sensitive and allowed repeated readings of the same plate after various incubation time intervals. The method provides further advantages of (1) elimination of the need for expensive and hazardous radioactive materials, (2) relative ease and rapidity in which experiments may be performed and quantitated, (3) sensitivity and reproducibility, and (4) versatility of the assay for measuring cytotoxicity of either erythrocyte or nucleated target cells.

Animals↗
PubMed1986

Glycosaminoglycans enhance complement hemolytic efficiency: theoretical considerations for GAG-complement-saliva interactions.

When human serum is diluted and pre-incubated at 37 degrees C in low ionic strength buffer (LIS, u = 0.07; made iso-osmotic with dextrose), a spontaneous activation of complement (C) is observed as determined by C4 and C3 electrophoretic conversion. In this paper it is postulated that most species of glycosaminoglycans (GAG) restricted non-specific fluid phase complement consumption induced by LIS, an effect which conserved complement and thereby enhanced the subsequent residual serum C mediated hemolytic activity. The capacity of glycosaminoglycans, to have modulated the hemolytic activity at low ionic strength, depended on the charge of the GAG species tested. In general, the GAG regulatory effects may have been due to GAG mediated restriction of spontaneous non-specific fluid phase C1 autoactivation, and/or restriction of activated C1 activity. Such effects would result in the subsequent reduction of the spontaneous fluid phase C4 and C3 consumption. Although the precise mechanisms responsible for the effects were not identified, it is speculated that the potentiation of C1 inhibitor function and direct effects on C1 might be involved. Overall, the relative specific activities of the glycosaminoglycans, on a weight basis, in mediating the fluid phase C regulatory effect were heparin greater than dermatan sulfate greater than chondroitin-6-sulfate greater than chondroitin-4-sulfate greater than hyaluronic acid and keratan sulfate. When much higher concns of heparin (greater than or equal 0.2 micrograms/ml) were used, complement mediated lysis of EA was inhibited, probably due to the direct inhibition of C1, even C1 which may have bound to the sensitized erythrocytes (EA). Results similar to that of heparin were obtained using greater than 1 mg/ml of dermatan sulfate or dextran sulfate. In contrast, pre-incubation of human serum in LIS with high concns (up to 10 mg/ml) of hyaluronic acid or chondroitin-4-sulfate, which are much less charged, continued to result only in the restriction of hemolytically non-specific (fluid phase) C consumption, resulting in a higher residual complement hemolytic activity. A theory is developed that the binding of polyionic GAG to C1 and to C1 INH may provide a charged local environment which simulates a relatively higher ionic strength. Chemical degradation of hyaluronic acid or chondroitin-4 or -6 sulfate resulted in lowering of this C modulating effect, indicative of the importance of the structural integrity of these charged glycosaminoglycans.(ABSTRACT TRUNCATED AT 400 WORDS)

Complement Activation↗
PubMed1985

Unusual complement-mediated hemolytic kinetics at low ionic strength.

The dilution of human serum in relatively low ionic strength buffer (mu = 0.070) caused the spontaneous activation of C1 and a limited activation of C4 and C3 in the fluid phase. The unusual degree of activation of the complement system in the fluid phase suggested that the optimal functions of complement-regulatory systems such as C1 inhibitor might be reduced. As a function of the time of preincubation (PI) of diluted serum at 37 degrees C, under low ionic strength conditions, an unusual complement-mediated hemolytic kinetic pattern was observed upon adding sensitized erythrocytes (EA). For a 1:36 dilution of human serum, there was an initial progressive decrease in complement hemolytic activity (from 3 to 20 min PI, phase I), followed by an apparent functional reversal (increase) in hemolytic activity (20-50 min PI, phase II) and finally a gradual irreversible depletion of the hemolytic activity (after 50 min PI, phase III). This hemolytic pattern could only be adequately demonstrated using a kinetic assay which followed the course of lysis of EA in the presence of low dilutions of human serum as a complement source. Others might have missed this observation due to the use of end-point titration methods which required the use of relatively elevated serum dilutions at the time of EA addition. Mechanisms which governed the variations in hemolytic activity at low ionic strength were not clear. Speculatively, partial consumption of early complement components, generation of free C1q and generation of complement fragments might have accounted for the initial decrease in the hemolytic activity observed in phase I. The apparent functional reversal of hemolytic activity observed in phase II might have involved a critical depletion of C1 inhibitor which occurred secondary to C1 inhibitor binding to C1 (activated by low ionic strength effects) and to the C1 activated at the time of EA addition. Without sufficient regulation, a rapid unrestricted C1-mediated complement activation could have occurred, which resulted in a rapid deposition of complement on the EA. Finally, prolonged exposure of serum to low ionic strength effects appeared to induce a significant complement consumption, which caused a time-dependent irreversible depletion of complement hemolytic activity (phase III). Excess exogenous C1 inhibitor, when co-incubated with diluted serum at low ionic strength, reversed the time-dependent effects of low ionic strength and enhanced the subsequent specific complement-mediated hemolytic activity as compared to controls.(ABSTRACT TRUNCATED AT 400 WORDS)

Complement Activation↗
PubMed1985

Hyaluronic acid-complement interactions--II. Role of divalent cations and gelatin.

Native hyaluronic acid (HA) is reported to be a weak anticomplementary agent. However, the normal buffer systems used for complement tests incorporate gelatin, Ca2+ and Mg2+, which may bind to HA, influence its conformation and interfere with its anticomplementary reactions with complement components such as Cl. In this study, metal ions (Ca2+ and Mg2+), gelatin and fibronectin appeared to react with native HA preparations and block their anticomplementary effects on Cl. In previous studies, we obtained evidence for a relationship between reversible heat-induced HA conformational changes and a subsequent reversible increase in anticomplementary activity. The anticomplementary activity of heat-treated HA preparations was also reduced by gelatin.

Binding Sites↗
PubMed1985

Hyaluronic acid-complement interactions--I. Reversible heat-induced anticomplementary activity.

The in vitro interaction of hyaluronic acid (HA) with complement (C) classical-pathway activity has been investigated. It was found that native HA, even at a high concn (greater than 3 mg/ml), has a relatively weak anticomplementary activity. However, we report here that native HA can be reversibly altered by heat treatment such that C-inhibitory properties are manifested. We have determined in this study that a potent C-inhibitory activity can be obtained if HA solutions are thermally treated (100 degrees C), and stabilized by prompt freezing with prompt thawing just prior to the interaction with human serum complement. Several investigators have proposed that the intermolecular-associated strands of HA undergo a reversible decoupling upon thermal treatment and this decoupled state of HA can be semi-stabilized by quickly cooling the sample. This heat-treated HA strongly inhibits C1 as well as classical-pathway-mediated C3 conversion. However, if heat-treated HA samples are not stabilized but, rather, slowly cooled after heating or if heated HA samples are snapfrozen and then slowly thawed, the anticomplementary activity is gradually lost. Interestingly, the activity for this same sample can be regenerated by retreatment of the same sample with heat followed by low-temp stabilization, indicating the reversibility of the physical state of HA responsible for the anticomplementary effect. Since no detectable molecular degradation of thermally-treated HA was found, it was assumed that a heat-induced physical transition of HA (decoupled state) was responsible for the C-inhibitory effect.

Chromatography, Gel↗